Red cells of individuals with sickle cell anemia have various abnormalities of membran structure and function which ultimately lead to an irreversible change in the cell membrane, producing the characteristic sickled morphology. Irreversibly sickled cells have abnormal flexibility and fragility, with altered rheological properties in circulation. The cause of the irreversible alteration is not known, but must be related to membrane skeletal protein interactions. Specific aims of the proposed project are to identify the abnormal interaction between membrane skeletal change becomes irreversible. The following approaches and methodologies will be used: 1) Discocytes, dense discosytes and ISCs will be isolated from HbSS individuals by density gradient centrifugation, and red cells from HbSS individuals will be caused to sickle irreversibly by deoxygenation, in vitro and separated from unsickled cells by gradient centrifugation; 2) Membrane skeletal proteins, spectrin, actin and bands 2.1 and 4.1 will be isolated, rebound to depleted membranes of each cell type, or to each other, ane their binding constants and numbers of binding sites determined; 3) Bifunctional cross-linking reagents will be used to search for newly formed interactions among skeletal and integral membrane proteins; 4) hybrids - normal ghosts contianing HbS instead of HbA will be prepared and used to study the effects of Hb binding, ions and metabolites on the irreversible interaction, By these methods, the site of the defect in the membrane skeleton causing the irreversible shape change may be identified.